ORCID Profile
0000-0002-4847-9263
Current Organisations
Macquarie University
,
Duke University
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Publisher: Wiley
Date: 21-03-2014
DOI: 10.1002/BRB3.225
Publisher: Elsevier BV
Date: 11-2015
Publisher: Springer Science and Business Media LLC
Date: 11-11-2012
DOI: 10.1038/NN.3255
Publisher: Wiley
Date: 17-08-2020
Publisher: BMJ
Date: 05-11-2020
Abstract: Since the first report of CHCHD10 gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in CHCHD10 has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia. Whole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in CHCHD10 . CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology. No causal, novel or disease-associated variants in CHCHD10 were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease. Genetic variation in CHCHD10 is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis.
Publisher: The Royal Society
Date: 10-2017
DOI: 10.1098/RSOB.170058
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive weakness, paralysis and muscle loss often resulting in patient death within 3–5 years of diagnosis. Recently, we identified disease-linked mutations in the CCNF gene, which encodes the cyclin F protein, in cohorts of patients with familial and sporadic ALS and frontotemporal dementia (FTD) (Williams KL et al . 2016 Nat. Commun. 7 , 11253. ( doi:10.1038/ncomms11253 )). Cyclin F is a part of a Skp1-Cul-F-box (SCF) E3 ubiquitin-protein ligase complex and is responsible for ubiquitylating proteins for degradation by the proteasome. In this study, we investigated the phosphorylation status of cyclin F and the effect of the serine to glycine substitution at site 621 (S621G) on E3 ligase activity. This specific mutation (S621G) was found in a multi-generational Australian family with ALS/FTD. We identified seven phosphorylation sites on cyclin F, of which five are newly reported including Ser621. These phosphorylation sites were mostly identified within the PEST (proline, glutamic acid, serine and threonine) sequence located at the C-terminus of cyclin F. Additionally, we determined that casein kinase II (CK2) can phosphorylate Ser621 and thereby regulate the E3 ligase activity of the SCF (cyclin F) complex. Furthermore, the S621G mutation in cyclin F prevents phosphorylation by CK2 and confers elevated Lys48-ubiquitylation activity, a hallmark of ALS/FTD pathology. These findings highlight the importance of phosphorylation in regulating the activity of the SCF (cyclin F) E3 ligase complex that can affect downstream processes and may lead to defective motor neuron development, neuron degeneration and ultimately ALS and FTD.
Publisher: Oxford University Press (OUP)
Date: 02-2018
DOI: 10.1093/HMG/DDY041
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder and mutations in superoxide dismutase 1 (SOD1) account for 20% of familial ALS cases. The aetiology of ALS remains unclear, but protein misfolding, endoplasmic reticulum (ER) stress and neuronal apoptosis are implicated. We previously established that protein disulphide isomerase (PDIA1) is protective against ER stress and apoptosis in neuronal cells expressing mutant SOD1, and recently mutations in PDIA1 and related PDI family member endoplasmic reticulum protein 57 (ERp57/PDIA3), were associated with ALS. Here, we examined whether ERp57 is also protective against mutant SOD1 or whether distinct specificity exists amongst in idual PDI family members. Neuronal cells co-expressing SOD1 and ERp57 were examined for inclusion formation, ER stress, ubiquitin proteasome system (UPS) dysfunction and apoptosis. Over-expression of ERp57 inhibited inclusion formation, ER stress, UPS dysfunction and apoptosis, whereas silencing of ERp57 expression enhanced mutant SOD1 inclusion formation, ER stress and toxicity, indicating a protective role for ERp57 against SOD1 misfolding. ERp57 also inhibited the formation of mutant SOD1 inclusions and apoptosis in primary cortical neurons, thus confirming results obtained from cell lines. ERp57 partially co-localized with TAR DNA-binding protein-43 (TDP-43)-positive inclusions in spinal cords from sporadic ALS patients, thus linking ERp57 to protein misfolding in human sporadic disease. Our results therefore imply that ERp57 has a protective role against pathological events induced by mutant SOD1 and they link ERp57 to the misfolding of TDP-43. This study therefore has implications for the design of novel therapeutics based on the activities of the PDI family of proteins.
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C7CC09257A
Abstract: A superior biocompatible spherical nucleic acid (SNA) conjugate was fabricated by grafting siRNA onto the surface of a core composed of a spherical DNA nanostructure that we have termed DNA nanoclew (DC).
Publisher: Elsevier BV
Date: 09-2016
Publisher: Elsevier BV
Date: 10-2010
DOI: 10.1016/J.BIOCEL.2010.06.016
Abstract: Transactive response DNA binding protein 43 kDa (TDP-43) is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs). TDP-43 serves multiple functions with roles in transcriptional regulation, pre-mRNA splicing and translational regulation. TDP-43 is also crucial for embryonic development with increasing evidence indirectly implicating its involvement in other cellular processes including microRNA biogenesis, apoptosis and cell ision. The role of TDP-43 in neurodegeneration has been actively studied since identification as a major component of the ubiquitinated inclusions seen in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 pathology has also been identified in several other neurodegenerative diseases. These disorders are collectively referred to as TDP-43 proteinopathies. The identification of rare TDP-43 mutations in sporadic and familial forms of ALS and FTLD suggests TDP-43 plays an important pathogenic role, rather than merely being a marker of the disease.
Publisher: Elsevier BV
Date: 08-2017
DOI: 10.1016/J.BIOCEL.2017.06.011
Abstract: Cyclin F, encoded by CCNF, is the substrate recognition component of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCF
Publisher: Wiley
Date: 2008
DOI: 10.1002/BIP.21019
Abstract: Understanding the nature of binding of polycationic dendrimers to DNA provides useful information on their role in gene delivery. In the present study, we have characterized the interaction of several peptide-based polycationic dendrimers with salmon sperm DNA using isothermal titration calorimetry. The dendrimers consisted of the cell penetrating peptide TAT, a nuclear localization signal peptide and dendritic polylysine. The binding affinity and thermodynamic parameters were found to increase as the number of positive charges on the dendrimer increased, indicating that ionic interactions were the major binding forces between the two molecules. The effect of acidic pH (3.2) compared to a more neutral pH (7.2) was also examined. The binding affinity was stronger at the lower pH but precipitation of the complex was more prominent at pH 7.2 which was shown by large enthalpies. The results indicate that our dendrimers are forming stable complexes with DNA.
Publisher: Springer Science and Business Media LLC
Date: 27-05-2015
DOI: 10.1007/S12640-015-9532-1
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by the progressive degeneration of brain and spinal cord motor neurons. Ubiquitin-proteasome system (UPS) dysfunction and oxidative stress have been implicated in ALS pathogenesis. However, it is unknown whether the defects in these pathways extend to non-neuronal tissues such as fibroblasts. Fibroblasts, unlike neuronal tissue, are readily available and may hold potential for short-term, rapid diagnostic and prognostic purposes. We investigated whether primary skin fibroblasts from ALS patients share, or can be manipulated to develop, functional and pathological abnormalities seen in affected neuronal cells. We inhibited UPS function and induced oxidative stress in the fibroblasts and found that ALS-related cellular changes, such as aggregate formation and ubiquitination of ALS-associated proteins (TDP-43 and ubiquilin 2), can be reproduced in these cells. Higher levels of TDP-43 ubiquitination, as evident by colocalization between TDP-43 and ubiquitin, were found in all six ALS cases compared to controls following extracellular insults. In contrast, colocalization between ubiquilin 2 and ubiquitin was not markedly different between ALS cases and control. A UPS reporter assay revealed UPS abnormalities in patient fibroblasts. Despite the presence of ALS-related cellular changes in the patient fibroblasts, no elevated toxicity was observed. This suggests that aggregate formation and colocalization of ALS-associated proteins may be insufficient alone to confer toxicity in fibroblasts used in the present study. Chronic exposure to ALS-linked stresses and the ALS-linked cellular pathologies may be necessary to breach an unknown threshold that triggers cell death.
Publisher: Wiley
Date: 02-08-2021
DOI: 10.1111/NAN.12749
Abstract: Splicing factor proline and glutamine rich (SFPQ) is an RNA–DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case–control cohorts. We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA‐sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ‐positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.
Publisher: Informa Healthcare
Date: 03-2010
DOI: 10.1517/17425240903512931
Abstract: Non-viral gene delivery for the treatment of genetic and non-genetic diseases has been under investigation for several decades, but there has been very little application in patients because of poor gene expression and toxicity. As gene delivery almost invariably involves endocytosis, many of its limitations are related to compartmentalisation of the transgene within the endosomes. Gene expression enhancers have become an essential part of manipulating endosomal release, as well as protecting transgene from intracellular degradation. However, disruption of the endosomes can also release proteases that have been shown to activate apoptotic pathways. An understanding of the role that endosomal release plays in the toxicity of gene delivery vehicles will help identify new approaches to minimise adverse effects while enhancing non-viral gene expression. The future of non-viral gene therapy needs to identify new approaches that limit endosome-induced toxicity while enhancing expression so that a pharmacological response can be reliably observed in vivo.
Publisher: Wiley
Date: 2008
DOI: 10.1002/CNE.21616
Publisher: Informa UK Limited
Date: 16-03-2012
DOI: 10.3109/17482968.2012.662690
Abstract: Phenotypic variation in amyotrophic lateral sclerosis (ALS) is common, and one atypical form is the flail arm variant (FAV). Some classic ALS patients carry TARDBP mutations, and so we sought to establish whether TARDBP mutations are also present in the FAV of ALS. Mutation analysis of TARDBP, the gene encoding TDP-43, was performed in cohorts of classic and FAV ALS patients. An analysis of mutation effects was performed in patient fibroblasts. Results showed that a novel heterozygous in-frame insertion/deletion (indel), c.1158_1159delAT c.1158_1159insCACCAACC, was identified in a highly conserved region encoding the glycine-rich area of TDP-43 in a patient with FAV. This indel was confirmed in the proband's mother, an obligate carrier, and was absent from 480 ethnically-matched control in iduals. Transcription of the mutant allele was confirmed. Under induced stress, indel-mutant fibroblasts showed a loss of normal nuclear TDP-43 immunoreactivity and formation of cytoplasmic inclusions of TDP-43, consistent with features seen in affected neurons. In conclusion, TARDBP missense mutations have previously been reported in classic ALS and frontotemporal lobar degeneration. The identification of a TARDBP indel mutation in a patient with FAV extends the spectrum of mutations and further supports the role of TDP-43 in a range of neurodegenerative phenotypes.
Publisher: Elsevier BV
Date: 06-2022
DOI: 10.1016/J.NBD.2022.105673
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of upper and lower motor neurons in the brain and spinal cord. ALS and frontotemporal dementia (FTD) are overlapping diseases with shared pathological features. Affected neurons of people with ALS and FTD typically contain ubiquitin-immunoreactive inclusions, of which TDP-43 (Tar DNA-binding protein of 43 kDa) is a major component. However, what triggers the formation of these abnormal TDP-43 inclusions is unclear. Previously, we identified CCNF mutations in cohorts of familial and sporadic cases of ALS and FTD. CCNF encodes cyclin F, the substrate-binding component of a multiprotein E3 ubiquitin ligase complex that ubiquitylates and subsequently directs a set of protein substrates for proteasomal degradation. Here, we explored the relationship between cyclin F and TDP-43. We used a series of complementary biochemical approaches including immunoprecipitations, in vitro ubiquitylation assays, immunofluorescence imaging and immunocytochemistry. Unpaired student t-tests were used to determine statistical significance of the results. In this study, we demonstrate that that the SCF In summary, this study reports a direct ubiquitylation mechanism for TDP-43, revealing important insights into the regulation of cyclin F-mediated TDP-43 turnover and clues towards understanding the molecular origins of the ubiquitylated TDP-43 inclusions that are the hallmark pathological feature in ALS and FTD.
Publisher: MDPI AG
Date: 02-09-2020
DOI: 10.3390/CELLS9092018
Abstract: The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer’s disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch cl . We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.
Publisher: Wiley
Date: 10-2009
DOI: 10.1002/CNE.22111
Publisher: Oxford University Press (OUP)
Date: 17-03-2021
DOI: 10.1093/HMG/DDAB073
Abstract: Previously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Hallmark features of these diseases include the build-up of insoluble protein aggregates as well as the mislocalization of proteins such as transactive response DNA binding protein 43 kDa (TDP-43). In recent years, the dysregulation of SFPQ (splicing factor proline and glutamine rich) has also emerged as a pathological hallmark of ALS/FTD. CCNF encodes for the protein cyclin F, a substrate recognition component of an E3 ubiquitin ligase. We have previously shown that ALS/FTD-linked mutations in CCNF cause disruptions to overall protein homeostasis that leads to a build-up of K48-linked ubiquitylated proteins as well as defects in autophagic machinery. To investigate further processes that may be affected by cyclin F, we used a protein-proximity ligation method, known as Biotin Identification (BioID), standard immunoprecipitations and mass spectrometry to identify novel interaction partners of cyclin F and infer further process that may be affected by the ALS/FTD-causing mutation. Results demonstrate that cyclin F closely associates with proteins involved with RNA metabolism as well as a number of RNA-binding proteins previously linked to ALS/FTD, including SFPQ. Notably, the overexpression of cyclin F(S621G) led to the aggregation and altered subcellular distribution of SFPQ in human embryonic kidney (HEK293) cells, while leading to altered degradation in primary neurons. Overall, our data links ALS/FTD-causing mutations in CCNF to converging pathological features of ALS/FTD and provides a link between defective protein degradation systems and the pathological accumulation of a protein involved in RNA processing and metabolism.
Publisher: Society for Neuroscience
Date: 22-01-2020
DOI: 10.1523/JNEUROSCI.1306-19.2019
Abstract: ACh is a signaling molecule in the mammalian CNS, with well-documented influence over cognition and behavior. However, the nature of cholinergic signaling in the brain remains controversial, with ongoing debates focused on the spatial and temporal resolution of ACh activity. Generally, opposing views have embraced a dichotomy between transmission as slow and volume-mediated versus fast and synaptic. Here, we provide the perspective that ACh, like most other neurotransmitters, exhibits both fast and slow modes that are strongly determined by the anatomy of cholinergic fibers, the distribution and the signaling mechanisms of receptor subtypes, and the dynamics of ACh hydrolysis. Current methodological approaches remain limited in their ability to provide detailed analyses of these underlying factors. However, we believe that the continued development of novel technologies in combination with a more nuanced view of cholinergic activity will open critical new avenues to a better understanding of ACh in the brain. Dual Perspectives Companion Paper: Forebrain Cholinergic Signaling: Wired and Phasic, Not Tonic, and Causing Behavior, by Martin Sarter and Cindy Lustig
Publisher: Oxford University Press (OUP)
Date: 19-11-2015
DOI: 10.1093/HMG/DDU578
Abstract: Cytosolic accumulation of TAR DNA binding protein 43 (TDP-43) is a major neuropathological feature of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the mechanisms involved in TDP-43 accumulation remain largely unknown. Previously, we reported that inhibitors of cyclin-dependent kinases (CDKs) prevented cytosolic stress granule accumulation of TDP-43, correlating with depletion of heterogeneous ribonucleoprotein (hnRNP) K from stress granules. In the present study, we further investigated the relationship between TDP-43 and hnRNP K and their control by CDKs. Inhibition of CDK2 abrogated the accumulation of TDP-43 into stress granules. Phosphorylated CDK2 co-localized with accumulated TDP-43 and phosphorylated hnRNP K in stress granules. Inhibition of CDK2 phosphorylation blocked phosphorylation of hnRNP K, preventing its incorporation into stress granules. Due to interaction between hnRNP K with TDP-43, the loss of hnRNP K from stress granules prevented accumulation of TDP-43. Mutation of Ser216 and Ser284 phosphorylation sites on hnRNP K inhibited hnRNP K- and TDP-43-positive stress granule formation in transfected cells. The interaction between hnRNP K and TDP-43 was further confirmed by the loss of TDP-43 accumulation following siRNA-mediated inhibition of hnRNP K expression. A substantial decrease of CDK2 and hnRNP K expression in spinal cord motor neurons in ALS patients demonstrates a potential key role for these proteins in ALS and TDP-43 accumulation, indicating that further investigation of the association between hnRNP K and TDP-43 is warranted. Understanding how kinase activity modulates TDP-43 accumulation may provide new pharmacological targets for disease intervention.
Publisher: Elsevier BV
Date: 09-2010
DOI: 10.1016/J.BIOCEL.2010.06.003
Abstract: The fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene was initially identified as a component of a fusion pro-oncogene resulting from a chromosomal translocation seen in liposarcomas. FUS/TLS belongs to a sub-family of RNA binding proteins, encoding an N-terminal serine-tyrosine-glycine-glutamine (SYGQ) region, an RNA recognition motif (RRM) flanked by glycine rich (G-rich) regions, a cysteine(2)/cysteine(2) zinc finger motif and multiple RGG repeats. The FUS/TLS protein interacts with RNA, single stranded DNA and double stranded DNA, and is involved in unique functions in mRNA processing and transport, transcriptional regulation and maintenance of genomic stability. Recently, several mutations in this gene have been found in amyotrophic lateral sclerosis (ALS) patients. The mutant forms of FUS/TLS exhibit similar pathology to other ALS causative genes, including aberrant cytoplasmic inclusions and an increased FUS/TLS cytoplasmic to nuclear ratio. The FUS/TLS mutations identified in ALS patients suggests that altered RNA metabolism may play a role in ALS pathogenesis.
Publisher: Springer Science and Business Media LLC
Date: 09-09-2020
DOI: 10.1186/S13024-020-00386-4
Abstract: Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response. We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or H 2 O 2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage. We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm. This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.NEUROBIOLAGING.2012.05.008
Abstract: Amyotrophic lateral sclerosis (ALS) shows clinical and pathological overlap with frontotemporal dementia that includes the presence of hallmark ubiquitinated inclusions in affected neurons. Mutations in UBQLN2, which encodes ubiquilin 2, were recently identified in X-linked juvenile and adult-onset ALS and ALS/dementia. As part of an established exome sequencing program to identify disease genes in familial ALS, we identified a novel missense UBQLN2 mutation (c.1460C>T, p.T487I) in 2 apparently unrelated multigenerational ALS families with no evidence of frontotemporal dementia. This mutation segregated with the disease and was absent in 820 healthy controls and all public single nucleotide polymorphism databases. The UBQLN2 p.T487I mutation substitutes a highly conserved residue and is located immediately upstream of a PXX region where all previous mutations have been identified. Immunostaining of spinal cord from a patient with UBQLN2 p.T487I mutation showed colocalization of ubiquilin 2 with ubiquitin in all neuronal inclusions examined and frequent colocalization with TAR DNA-binding protein 43 (TDP-43) and fused in sarcoma protein (FUS). To examine ubiquilin 2 pathology in broader ALS, we showed that ubiquilin 2 pathology also extends to ALS with a FUS mutation. These data further support the importance of ubiquilin 2 in the pathogenesis of ALS.
Publisher: Elsevier BV
Date: 2020
Publisher: Springer Science and Business Media LLC
Date: 04-12-2019
DOI: 10.1007/S00018-019-03394-W
Abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases that are related genetically and pathologically. Mutations in the UBQLN2 gene, encoding the ubiquitin-like protein ubiquilin2, are associated with familial ALS/FTD, but the pathophysiological mechanisms remain unclear. Here, we demonstrate that ALS/FTD UBQLN2 mutants P497H and P506T inhibit protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in neuronal cells. In addition, we observed that Sec31-positive ER exit sites are clustered in UBQLN2
Publisher: Oxford University Press (OUP)
Date: 09-03-2017
DOI: 10.1093/HMG/DDX093
Abstract: TAR DNA binding protein 43 (TDP-43) is a major disease-associated protein involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Our previous studies found a direct association between TDP-43 and heterogeneous nuclear ribonucleoprotein K (hnRNP K). In this study, utilizing ALS patient fibroblasts harboring a TDP-43M337V mutation and NSC-34 motor neuronal cell line expressing TDP-43Q331K mutation, we show that hnRNP K expression is impaired in urea soluble extracts from mutant TDP-43 cell models. This was confirmed in vivo using TDP-43Q331K and inducible TDP-43A315T murine ALS models. We further investigated the potential pathological effects of mutant TDP-43-mediated changes to hnRNP K metabolism by RNA binding immunoprecipitation analysis. hnRNP K protein was bound to antioxidant NFE2L2 transcripts encoding Nrf2 antioxidant transcription factor, with greater enrichment in TDP-43M337V patient fibroblasts compared to healthy controls. Subsequent gene expression profiling revealed an increase in downstream antioxidant transcript expression of Nrf2 signaling in the spinal cord of TDP-43Q331K mice compared to control counterparts, yet the corresponding protein expression was not up-regulated in transgenic mice. Despite the elevated expression of antioxidant transcripts, we observed impaired levels of glutathione (downstream Nrf2 antioxidant) in TDP-43M337V patient fibroblasts and astrocyte cultures from TDP-43Q331K mice, indicative of elevated oxidative stress and failure of some upregulated antioxidant genes to be translated into protein. Our findings indicate that further exploration of the interplay between hnRNP K (or other hnRNPs) and Nrf2-mediated antioxidant signaling is warranted and may be an important driver for motor neuron degeneration in ALS.
Publisher: Springer Science and Business Media LLC
Date: 27-05-2023
DOI: 10.1007/S12035-023-03355-2
Abstract: Amyotrophic lateral sclerosis (ALS)- and frontotemporal dementia (FTD)-linked mutations in CCNF have been shown to cause dysregulation to protein homeostasis. CCNF encodes for cyclin F, which is part of the cyclin F-E3 ligase complex SCF cyclinF known to ubiquitylate substrates for proteasomal degradation. In this study, we identified a function of cyclin F to regulate substrate solubility and show how cyclin F mechanistically underlies ALS and FTD disease pathogenesis. We demonstrated that ALS and FTD-associated protein sequestosome-1 62 (p62) was a canonical substrate of cyclin F which was ubiquitylated by the SCF cyclinF complex. We found that SCF cyclin F ubiquitylated p62 at lysine(K)281, and that K281 regulated the propensity of p62 to aggregate. Further, cyclin F expression promoted the aggregation of p62 into the insoluble fraction, which corresponded to an increased number of p62 foci. Notably, ALS and FTD-linked mutant cyclin F p.S621G aberrantly ubiquitylated p62, dysregulated p62 solubility in neuronal-like cells, patient-derived fibroblasts and induced pluripotent stem cells and dysregulated p62 foci formation. Consistently, motor neurons from patient spinal cord tissue exhibited increased p62 ubiquitylation. We suggest that the p.S621G mutation impairs the functions of cyclin F to promote p62 foci formation and shift p62 into the insoluble fraction, which may be associated to aberrant mutant cyclin F-mediated ubiquitylation of p62. Given that p62 dysregulation is common across the ALS and FTD spectrum, our study provides insights into p62 regulation and demonstrates that ALS and FTD-linked cyclin F mutant p.S621G can drive p62 pathogenesis associated with ALS and FTD.
Publisher: Frontiers Media SA
Date: 27-04-2021
DOI: 10.3389/FNMOL.2021.627740
Abstract: The past decade has seen a rapid acceleration in the discovery of new genetic causes of ALS, with more than 20 putative ALS-causing genes now cited. These genes encode proteins that cover a erse range of molecular functions, including free radical scavenging (e.g., SOD1), regulation of RNA homeostasis (e.g., TDP-43 and FUS), and protein degradation through the ubiquitin-proteasome system (e.g., ubiquilin-2 and cyclin F) and autophagy (TBK1 and sequestosome-1 62). It is likely that the various initial triggers of disease (either genetic, environmental and/or gene-environment interaction) must converge upon a common set of molecular pathways that underlie ALS pathogenesis. Given the complexity, it is not surprising that a catalog of molecular pathways and proteostasis dysfunctions have been linked to ALS. One of the challenges in ALS research is determining, at the early stage of discovery, whether a new gene mutation is indeed disease-specific, and if it is linked to signaling pathways that trigger neuronal cell death. We have established a proof-of-concept proteogenomic workflow to assess new gene mutations, using CCNF (cyclin F) as an ex le, in cell culture models to screen whether potential gene candidates fit the criteria of activating apoptosis. This can provide an informative and time-efficient output that can be extended further for validation in a variety of in vitro and in vivo models and/or for mechanistic studies. As a proof-of-concept, we expressed cyclin F mutations (K97R, S195R, S509P, R574Q, S621G) in HEK293 cells for label-free quantitative proteomics that bioinformatically predicted activation of the neuronal cell death pathways, which was validated by immunoblot analysis. Proteomic analysis of induced pluripotent stem cells (iPSCs) derived from patient fibroblasts bearing the S621G mutation showed the same activation of these pathways providing compelling evidence for these candidate gene mutations to be strong candidates for further validation and mechanistic studies (such as E3 enzymatic activity assays, protein–protein and protein–substrate studies, and neuronal apoptosis and aberrant branching measurements in zebrafish). Our proteogenomics approach has great utility and provides a relatively high-throughput screening platform to explore candidate gene mutations for their propensity to cause neuronal cell death, which will guide a researcher for further experimental studies.
Publisher: ACM
Date: 30-04-2023
Publisher: Elsevier BV
Date: 10-2019
DOI: 10.1016/J.SCR.2019.101530
Abstract: Dermal fibroblasts from a 59 year old male patient with amyotrophic lateral sclerosis (symptomatic at the time of collection), attributed to a mutation in the cyclin F gene (CCNF
Publisher: Oxford University Press (OUP)
Date: 18-02-2014
DOI: 10.1093/HMG/DDU068
Publisher: Wiley
Date: 12-09-2010
Publisher: Wiley
Date: 2006
DOI: 10.1002/CNE.21096
Abstract: Cholinergic neuromodulation, a candidate mechanism for aspects of attention, is complex and is not well understood. Because structure constrains function, quantitative anatomy is an invaluable tool for reducing such a challenging problem. Our goal was to determine the extent to which m1 and m2 muscarinic acetylcholine receptors (mAChRs) are expressed by inhibitory vs. excitatory neurons in the early visual cortex. To this end, V1 and V2 of macaque monkeys were immunofluorescently labelled for gamma-aminobutyric acid (GABA) and either m1 or m2 mAChRs. Among the GABA-immunoreactive (ir) neurons, 61% in V1 and 63% in V2 were m1 AChR-ir, whereas 28% in V1 and 43% in V2 were m2 AChR-ir. In V1, both mAChRs were expressed by fewer than 10% of excitatory neurons. However, in V2, the population of mAChR-ir excitatory neurons was at least double that observed in V1. We also examined m1 and m2 AChR immunoreactivity in layers 2 and 3 of area V1 under the electron microscope and found evidence that GABAergic neurons localize mAChRs to the soma, whereas glutamatergic neurons expressed mAChRs more strongly in dendrites. Axon and terminal labelling was generally weak. These data represent the first quantitative anatomical study of m1 and m2 AChR expression in the cortex of any species. In addition, the increased expression in excitatory neurons across the V1/V2 border may provide a neural basis for the observation that attentional effects gain strength up through the visual pathway from area V1 through V2 to V4 and beyond.
Publisher: Springer Science and Business Media LLC
Date: 15-04-2016
DOI: 10.1038/NCOMMS11253
Abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping, fatal neurodegenerative disorders in which the molecular and pathogenic basis remains poorly understood. Ubiquitinated protein aggregates, of which TDP-43 is a major component, are a characteristic pathological feature of most ALS and FTD patients. Here we use genome-wide linkage analysis in a large ALS/FTD kindred to identify a novel disease locus on chromosome 16p13.3. Whole-exome sequencing identified a CCNF missense mutation at this locus. Interrogation of international cohorts identified additional novel CCNF variants in familial and sporadic ALS and FTD. Enrichment of rare protein-altering CCNF variants was evident in a large sporadic ALS replication cohort. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase complex (SCF Cyclin F ). Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCF Cyclin F substrate. This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.
Publisher: Elsevier BV
Date: 05-2014
DOI: 10.1016/J.BIOCEL.2014.02.018
Abstract: Ubiquilin 2, which is encoded by the UBQLN2 gene, plays a critical role in protein clearance pathways including the ubiquitin-proteasome system and autophagy. Ubiquilin 2 physically associates with ubiquitin ligases and proteasomes to mediate protein degradation. It also plays a role in the regulation of cell signalling and cell cycle progression, and association with cytoskeletal elements. Recent studies have revealed that ubiquilin 2 also plays a pathogenic role in neurodegenerative disease, including amyotrophic lateral sclerosis (ALS), and ALS-frontotemporal dementia (ALS-FTD). Rare UBQLN2 mutations cause a small subset of ALS and ALS-FTD cases. More widespread is the presence of ubiquilin 2 positive inclusions in the affected neurons of some familial and sporadic ALS and ALS-FTD patients. These discoveries have led to the hypothesis that perturbation in protein clearance, mediated by ubiquilin 2, is an important pathogenic mechanism in neurodegeneration.
Publisher: Elsevier BV
Date: 11-2007
Publisher: Cambridge University Press (CUP)
Date: 12-2004
DOI: 10.1017/S0140525X0425018X
Abstract: The cholinergic system is a good candidate for the role of determining the relative weight given in cortical information processing to new sensory information versus prior knowledge. We discuss the physiological data supporting this, and suggest that this Bayesian perspective can easily be reconciled with the dynamical framework proposed by Behrendt & Young (B& Y).
Publisher: Elsevier BV
Date: 2012
DOI: 10.1016/J.NEUROBIOLAGING.2011.09.023
Abstract: Mutations in the optineurin gene (OPTN) have been reported in rare familial and sporadic amyotrophic lateral sclerosis (ALS) cases. It is yet to be established whether mutations segregate with dominantly inherited familial ALS. We therefore performed mutation analysis in a cohort of 96 autosomal dominant ALS families. A novel heterozygous nonsynonymous variant (c.218C > T, S73L) was identified in one patient however, analysis in the extended pedigree demonstrated that this variant was inherited from an unaffected parent. The variant was absent in 480 control in iduals. The affected serine residue is highly conserved and its substitution is predicted to alter phosphorylation. Despite this, our evidence indicates that this variant is unlikely to play a pathogenic role in the disease. Cell and animal models will be required to functionally support the pathogenic role of OPTN mutations.
Publisher: Cold Spring Harbor Laboratory
Date: 27-02-2018
DOI: 10.1101/271924
Abstract: Release of the neuromodulator acetylcholine into cortical circuits supports cognition, although its precise role and mechanisms of action are not well-understood. Little is known about functional differences in cholinergic modulatory effects across cortical model systems, but anatomical evidence suggests that such differences likely exist because, for ex le, the expression of cholinergic receptors differs profoundly both within and between species. In the primary visual cortex (V1) of macaque monkeys, cholinergic receptors are strongly expressed by inhibitory interneurons. Here, we examine m1 muscarinic acetylcholine receptor expression by two subclasses of inhibitory interneurons—identified by their expression of the calcium-binding proteins calbindin and calretinin—in the middle temporal extrastriate area (MT) of the macaque. Using dual-immunofluorescence confocal microscopy, we find that the majority of calbindin-immunoreative neurons (55%) and only few calretinin-immunoreactive neurons (10%) express the m1 acetylcholine receptor. This differs from the pattern observed in V1 of the same species, lending further support to the notion that cholinergic modulation in cortex is tuned such that different cortical compartments will respond to acetylcholine release in different ways.
Publisher: Proceedings of the National Academy of Sciences
Date: 23-12-2019
Abstract: Biomedical research is at a critical juncture, with an aging population increasingly beset by chronic illness and prominent failures to translate research from “bench to bedside.” These challenges emerge on a background of increasing “silo-ing” of experiments (and experimenters)—many investigators produce and consume research conducted in 1, perhaps 2, species—and increasing pressure to reduce or eliminate research on so-called “higher” mammals. Such decisions to restrict species ersity in biomedical research have not been data-driven and increase the risk of translational failure. To illustrate this problem, we present a case study from neuroscience: cholinergic suppression in the cortex. In all mammals studied so far, acetylcholine reduces activity in some cortical neurons. Comparative anatomical studies have shown that the mechanism behind this suppression differs between species in a manner that would render drug treatments developed in nonprimate species entirely ineffective if applied to primates (including humans). Developing clinical interventions from basic research will always require translation, either between species (e.g., using a mouse model of a human disease) or within a species (using a subset of humans as a representative s le for all humans). We argue that successful translation will require that we 1) be data-driven in our selection of species for study 2) use (with careful attention to welfare) animals that minimize the translation gap to humans and 3) become agile at translation, by resisting the pressures to narrow our focus to a small number of organisms, instead using species ersity as an opportunity to practice translation.
Publisher: Springer Science and Business Media LLC
Date: 29-08-2018
DOI: 10.1007/S00018-017-2632-8
Abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43 however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCF
Publisher: Elsevier BV
Date: 09-2013
DOI: 10.1016/J.NEUROBIOLAGING.2013.04.003
Abstract: Mutations in PFN1, a gene encoding the actin monomer-binding protein profilin 1, were recently reported in 1% to 2% of familial amyotrophic lateral sclerosis (ALS) patients. In vitro functional studies suggested that PFN1 mutations lead to ubiquitin-positive inclusions and impairment of cytoskeletal pathways. In the present study, mutation analysis of PFN1 was performed in an Australian cohort of 110 ALS families and 715 sporadic ALS patients. No PFN1 mutations were identified in familial ALS patients. Two rare non-synonymous variants (E117D and E117G) were found in sporadic ALS patients at similar incidences to that reported in public SNP databases. Immunostaining of PFN1 in sporadic ALS and familial ALS patients, including those with mutations in SOD1, FUS, UBQLN2 and C9ORF72, found no PFN1-positive inclusions in spinal motor neurons. Our data suggest that PFN1 mutations and pathology are not common in an Australian ALS cohort of predominantly European ancestry.
Publisher: Elsevier BV
Date: 04-2009
DOI: 10.1016/J.JCONREL.2008.12.015
Abstract: A small library of carriers consisting of various combinations of the cell penetrating peptide TAT, the SV40 Large T protein nuclear localisation signal (NLS) and a cationic dendrimer of 7 lysine residues (DEN) was synthesised and each member was tested for its ability to deliver exogenous DNA to human HeLa cells. We found that the TAT peptide was essential, but not sufficient for efficient uptake of exogenous DNA. The addition of either NLS or DEN significantly enhanced uptake and expression with the most active carrier consisting of the TAT, NLS and DEN peptides. For those peptides that facilitated DNA uptake, the complexes were targeted to intracellular compartments that required incubation with a fusogenic agent such as chloroquine before gene expression was observed. However, our data suggest that chloroquine did not enhance expression solely by promoting endosomal release since a fusogenic peptide derived from the influenza virus haemagglutinin protein did not improve gene expression. Chloroquine was found to protect DNA from degradation and enhance transcription of DNA bound to the respective carriers. Our results demonstrate that multi-component peptide-based gene carriers can be designed to deliver exogenous DNA. The actions of lysosomotropic agents such as chloroquine reveal the multifactorial properties required for carriers used in non-viral gene delivery.
Publisher: Wiley
Date: 20-12-2018
DOI: 10.1002/CNE.24590
Publisher: Oxford University Press (OUP)
Date: 10-08-2017
DOI: 10.1093/HMG/DDX309
Publisher: S. Karger AG
Date: 2017
DOI: 10.1159/000481258
Abstract: b i Background: /i /b Mutations in the genes encoding the heterogeneous nuclear ribonucleoproteins hnRNPA1 and hnRNPA2/B1 have been reported in a multisystem proteinopathy that includes amyotrophic lateral sclerosis (ALS) and inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia. Mutations were also described in the prion-like domain of hnRNPA1 in patients with classic ALS. Another hnRNP protein, hnRNPA3, has been found to be associated with the ALS/frontotemporal dementia protein C9orf72. b i Objective: /i /b To further assess their role in ALS, we examined these hnRNPs in spinal cord tissue from sporadic (SALS) and familial ALS (FALS) patients, including i C9orf72 /i repeat expansion-positive patients, and controls. We also sought to determine the prevalence of i HNRNPA1 /i , i HNRNPA2B1, /i and i HNRNPA3 /i mutations in Australian ALS patients. b i Methods: /i /b Immunostaining was used to assess hnRNPs in ALS patient spinal cords. Mutation analysis of the i HNRNPA1 /i , i HNRNPA2B1, /i and i HNRNPA3 /i genes was performed in FALS and of their prion-like domains in SALS patients. b i Results: /i /b Immunostaining of spinal motor neurons of ALS patients with the i C9orf72 /i repeat expansion showed significant mislocalisation of hnRNPA3, and no differences in hnRNPA1 or A2/B1 localisation, compared to controls. No novel or known mutations were identified in i HNRNPA1 /i , i HNRNPA2B1, /i or i HNRNPA3 /i in Australian ALS patients. b i Conclusions: /i /b hnRNPA3 pathology was identified in motor neurons of ALS patients with i C9orf72 /i repeat expansions, implicating hnRNPA3 in the pathogenesis of i C9orf72 /i -linked ALS. hnRNPA3 warrants further investigation into the pathogenesis of ALS linked to i C9orf72 /i . This study also determined that i HNRNP /i mutations are not a common cause of FALS and SALS in Australia.
Publisher: Elsevier BV
Date: 12-2007
Location: United States of America
No related grants have been discovered for Shu Yang.